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| United States Patent |
5,631,348
|
|
Rosenblum
, et al.
|
May 20, 1997
|
Protein sequence of the plant toxin gelonin
Abstract
This invention relates to substantially purified gelonin, toxic fragments
thereof, the DNA sequences encoding gelonin and use of the DNA for
producing, by recombinant technology, gelonin, toxic fragments thereof and
fusion proteins. More specifically, the invention relates to the primary
amino acid sequence of gelonin, and of the DNA encoding said gelonin and
the production of synthetic gelonin and toxic fragments thereof.
| Inventors:
|
Rosenblum; Michael (Houston, TX);
Kohr; William J. (San Mateo, CA);
Aggarwal; Bharat (Houston, TX)
|
| Assignee:
|
The Research Development Foundation (Carson City, NV)
|
| Appl. No.:
|
254662 |
| Filed:
|
June 6, 1994 |
| Current U.S. Class: |
530/370; 536/23.6 |
| Intern'l Class: |
C07K 014/415; C12N 015/29 |
| Field of Search: |
536/23.6
530/370
435/172.3,252.3
|
References Cited
U.S. Patent Documents
| 4446240 | May., 1984 | Nerenberg | 436/542.
|
| 4810648 | Mar., 1989 | Stalker | 435/191.
|
| 4888415 | Dec., 1989 | Lambert et al. | 530/390.
|
| 4921802 | May., 1990 | Hall et al. | 435/172.
|
Other References
Lambert et al., Cancer Treat. Res., vol. 37, 1988, pp. 175-209.
Ronneberger, Dev. Biol. Stand., vol. 71, 1990, pp. 185-190.
Benoit et al., Curr. Eye Res., vol. 9 Suppl., 1990, pp. 201-205.
Roselli et al., J. Nuclear Med., vol. 30, 1989, pp. 672-682.
Dunbar, Two-Dimensional Electrophoresis and Immunological Techniques, 1987,
Plenum Press, New York, pp. 32-34.
Stirpe et al., J. Biol. Chem. v. 255, 1980, pp. 6947-6953.
Jacobs, et al., Nature v. 313, 1985, pp. 806-810.
Falasca et al., Biochem. J. v. 207, 1982, pp. 505-509.
Sivam et al., Cancer Res. v. 47, 1987, pp. 3169-3173.
Young et al., Science v. 222, 1983, pp. 778-782.
Toy, J. L., Clin. Exp. Immunol., 1983, 54, 1-13.
|
Primary Examiner: Ketter; James S.
Attorney, Agent or Firm: Adler; Benjamin Aaron
Parent Case Text
This is a continuation of application Ser. No. 08/119,899 filed on Sep. 10,
1993, now abandoned, which is a continuation of U.S. Ser. No. 07/908,959
filed Jul. 6, 1992, abandoned, which is a continuation of U.S. Ser. No.
07/567,220, filed Aug. 14, 1990, now abandoned.
Claims
What is claimed as new and is desired to be covered under Letters Patent
is:
1. Substantially pure gelonin toxin having the amino acid sequence:
##STR1##
or a fragment or derivative thereof, said fragment or derivatives or
having an activity which inhibits cellular protein synthesis but does not
bind to a cell surface receptor.
2. A DNA sequence of the formula:
__________________________________________________________________________
GGNYTNGAYA
CNGTNWSNTT
YWSNACNAAR
GGNGCNACNT
AYATHACNTA
YGTNAAYTTY
60
YTNAAYGARY
TNMGNGTNAA
RYTNAARCCN
GARGGNAAYW
SNCAYGGNAT
HCCNYTNYTN
120
MGNAARGGNG
AYGAYCCNGG
NAARTGYTTY
GTNYTNGTNG
CNYTNWSNAA
YGAYAAYGGN
180
CARYTNGCNG
ARATHGCNAT
HGAYGTNACN
WSNGTNTAYG
TNGTNGGNTA
YCARGTNMGN
240
AAYMGNWSNT
AYTTYTTYAA
RGAYGCNCCN
GAYGCNGCNT
AYGARGGNYT
NTTYAARAAY
300
ACNATHAARA
AYCCNYTNYT
NTTYGGNGGN
AARACNMGNY
TNCAYTTYGG
NGGNWSNTAY
360
CCNWSNYTNG
ARGGNGARAA
RGCNTAYMGN
GARACNACNG
AYYTNGGNAT
HGARCCNYTN
420
MGNATHGGNA
THAARAARYT
NGAYGARAAY
GCNATHGAYA
AYTAYAARCC
NACNGARATH
480
GCNWSNWSNY
TNYTNGTNGT
NATHCARATG
GTNWSNGARG
CNGCNMGNTT
YACNTTYATH
540
GARAAYCARA
THMGNAAYAA
YTTYCARCAR
MGNATHMGNC
CNGCNAAYAA
YACNATHWSN
600
YTNGARAAYA
ARTGGGGNAA
RYTNWSNTTY
CARATHMGNA
CNWSNGGNGC
NAAYGGNATG
660
TTYWSNGARG
CNGTNGARYT
NGARMGNGCN
AAYGGNAARA
ARTAYTAYGT
NACNGCNGTN
720
GAYCARGTNA
ARCCNAARAT
HGCNYTNYTN
AARTTYGTNG
AYAARGAYCC
NGAR 774
wherein R = A or G K = G or T N = any
Y = C or T M = A or C S = C or G
B = C, G, or T V = A, C, or G W = A or T
D = A, G, or T H = A, C, or T X = unknown
__________________________________________________________________________
or a fragment or derivative thereof, said fragment or derivative coding for
gelonin or for a polypeptide having an activity which inhibits cellular
protein synthesis but does not bind to a cell surface receptor.
Description
TECHNICAL FIELD
This invention relates to substantially purified gelonin, toxic fragments
thereof, the DNA sequences encoding gelonin and use of the DNA for
producing, by recombinant technology, gelonin, toxic fragments thereof and
fusion proteins. More specifically, the invention relates to the primary
amino acid sequence of gelonin, and of the DNA encoding said gelonin and
the production of synthetic gelonin and toxic fragments thereof.
BACKGROUND ART
A major challenge for the design of a drug for treatment of any disease is
specificity and efficacy. Various drugs available for the treatment of
cancer suffer from problems of this nature. The concept of targeting toxic
drugs selectively to certain tumors has been a subject of intense research
in the last few years (Thorpe (1985) Biol Clin Applications 84:475-512;
Moller ed. (1982) Immun. Rev. 62:1-215). Recently both monoclonal and
polyclonal antibodies, lectins, lymphokines and hormones which recognize
specific determinants on the surface of the tumor cell have been used as
carriers to deliver toxic agents into the cell, where the latter can exert
their cytotoxic potential (Blattler, et al. (1985) Biochemistry
24:1517-1524; Frankel, et al. (1985) J. Biol, Res, Modif, 4:437-446;
Reimann, et al. (1988) J. Clin. Invest. 82:129-138; Schwartz and Vale
(1988) Endocrinology 122:1695-1700; Scott, et al. (1987) J Natl. Cancer
Inst. 79:1163-1172; Singh, et al. (1989) Biol. Chem. 264:3089-3095;
Srinivasan, et al. (1985) FEBS Letters 192:113; Schwartz, et al. (1987)
Endocrinology 121:1454-1460). Toxic moieties thus far investigated with
these delivery agents include radionuclides (Ghose, et al. (1967) Br, Med,
J. 1:90-96), cytotoxic drugs commonly employed in cancer chemotherapy
(Thorp and Ross (1982) Immun. Rev, 62:119-157; Deweger, et al. (1982)
Immun. Rev. 62:29-45; Arnon and Sela (1982) Immun. Rev. 62:5-27; Pimm, et
al. (1982) Cancer Immun. Immunotherap, 12:125-134; Rowland and Axton
(1985) Cancer Immun. Immunotherap. 19:1-7) and proteins derived from
bacteria and plants such as diptheria or ricin (Jansen, et al. (1982)
Immun. Rev. 62:185-216; Raso (1982) Immun. Rev. 62:93-117. Vitetta, et al.
(1982) Immun. Rev, 62:159-183; Nelville and Youle (1982) Immun. Rev,
92:47-73; Thorpe, et al. (1981) Eur. J. Biochem. 116:447-454). A specific
molecule is designed by replacing the nonspecific B chain with an antibody
or a hormone.
Bacterial and plant toxins, such as diphtheria toxin (DT), Pseudomonas
aeruginosa toxin A, abrin, ricin, mistletoe, modeccin, and Shigella toxin,
are potent cytocidal agents due to their ability to disrupt a critical
cellular function. For instance, DT and ricin inhibit cellular protein
synthesis by inactivation of elongation factor-2 and inactivation of
ribosomal 60s subunits, respectively (Bacterial Toxins and Cell Membranes,
Eds. Jelajaszewicz and Wadstrom (1978) Academic Press, p. 291). These
toxins are extremely potent because they are enzymes and act catalytically
rather than stoichiometrically. The molecules of these toxins are composed
of an enzymatically active polypeptide chain or fragment, commonly called
"A" chain or fragment, linked to one or more polypeptide chains or
fragments, commonly called "B" chains or fragments, that bind the molecule
to the cell surface and enable the A chain to reach its site of action,
e.g., the cytosol, and carry out its disruptive function. The act of
gaining access to the cytosol is called variously "internalization",
"intoxication", or "translocation". These protein toxins belong to a class
bearing two chains referred to as A and B chains. The B chain has the
ability to bind to almost all cells whereas the cytotoxic activity is
exhibited by the A chain. It is believed that the A chain must be timely
liberated from the B chain-frequently by reduction of a disulfide bond-in
order to make the A chain functional. These natural toxins are generally
not selective for a given cell or tissue type because their B chains
recognize and bind to receptors that are present on a variety of cells.
The availability of a toxin molecule which is not cytotoxic to a variety of
cells when administered alone has been limited. Utilizing certain
naturally occurring single chain toxin molecules which do not themselves
bind to cell surface receptors and, therefore, are not normally
internalized by cells, has provided toxic molecules which are relatively
non-toxic to most, if not all, cells when administered alone. Such
naturally occurring single chain toxins known to date, include, but are
not limited to, pokeweed antiviral protein (Ramakrishnan and Houston
(1984) Cancer Res. 44:201-208), saponin (Thorpe, et al. (1985) J. Natl,
Cancer Inst. 75:151-159), and gelonin (Stirpe, et al (1980) J. Biol. Chem.
255:6947-6953). These proteins are nontoxic to cells in the free form, but
can inhibit protein synthesis once they gain entry into the cell. However,
the availability of these single chain toxins in substantially pure form
is limited due to the fact that they must be purified from plant sources
in which they occur in relatively low amounts and the reproducibility of
the concentration of the toxin in the plants is dependent upon plant
growth conditions and plant harvest conditions.
Gelonin is a single chain polypeptide isolated from seeds of a plant,
Gelonium multiforum, having a molecular weight of approximately
28,000-30,000 kd. Gelonin is a basic glycoprotein with an approximate
isoelectric point of 8.15 and contains mannose and glucosamine residues
(Falasca, et al. (1982) Biochem J, 207:505-509). In contrast to other
plant and bacterial toxins, this protein is not toxic to cells by itself,
but when delivered to cells through a carrier, it damages the 60s
ribosomal subunit. In vivo and in vitro biological data suggest that
gelonin is equivalent or superior to other plant toxins. In fact, the
results of a comparison of gelonin conjugates in vitro and in vivo with
other A chain conjugates indicated that gelonin had similar potency,
better selectivity, better tumor localization, and more significant
therapeutic effects (Sivan, et al (1987) Cancer Res, 47:3169-3173).
However, the availability of a reproducible, readily accessible supply of
gelonin from natural sources is limited. In addition, the purification of
gelonin from plant sources is difficult and the yield is very low.
Gelonin by itself has been shown to be abortifacient in mice and enhances
antibody dependent cell cytotoxicity (Yeung, et al (1988) Internatl. J.
Peptide Protein Res, 31:265-268).
Several investigators have utilized gelonin as a cytotoxic agent chemically
attached to monoclonal antibodies or to peptide hormone cellular targeting
ligands. However, chemical modification of gelonin and cellular targeting
moleties can reduce targeting efficiently and cytotoxic potential of
gelonin itself. Furthermore, natural sources of gelonin are subject to
variability in harvesting and plant growth which can affect gelonin
cytotoxic activity. The ability to produce a synthetic gelonin toxin,
chemically or utilizing recombinant technology, provides a plentiful,
reproducible source of the toxin.
SUMMARY OF THE INVENTION
The present invention provides substantially pure gelonin having the amino
acid sequence shown in FIG. 1. The present invention also provides the DNA
sequence for gelonin shown in FIG. 2. Utilization of the sequences of the
present invention to produce substantially pure gelonin in plentiful
amounts by recombinant technology provides abundant amounts of the toxin
which were not heretofore available from natural sources.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid sequence of gelonin.
FIG. 2 demonstrates the cDNA encoding for gelonin.
FIG. 3 demonstrates the homology of the gelonin amino acid sequence with
the sequence of trichosanthin, Ricin A chain, Agglutinin precursor
isolated from Castor bean and Abrin A chain.
FIG. 4 demonstrates the HPLC profile of CNBr fragments.
FIGS. 5A, 5B and 5C demonstrates the HPLC profile of (A) Lys-c, (B)
Staphyloccus protease, and (C) Hydroxylamine digests of gelonin.
FIGS. 6A, 6B, 6C and 6D demonstrates the hydrophobicity plots of gelonin
(A), trichosanthin (B), abrin (C), ricin (D), and agglutinin precursor (E)
.
DETAILED DESCRIPTION OF THE INVENTION
The term "substantially pure" when applied to the gelonin protein of the
present invention means that the polypeptide is essentially free of other
plant proteins normally associated with the gelonin in its natural state
and exhibiting reproducible electrophoretic or chromatographic response,
elution profiles, and toxic activity. The term "substantially pure" is not
meant to exclude artificial or synthetic mixtures of the gelonin protein
with other compounds.
Gelonin was purified from the seeds of the plant Gilonin multiforum by
techniques known to those of skill in the art. The amino acid sequence was
determined utilizing a modification of the Edman degradation method.
Samples of gelonin were applied to the reverse phase reaction chamber and
subjected to Edman degradation. The N-terminal of gelonin was found to be
heterogeneous (1/2 of the molecules of the protein were apparently one
amino acid shorter than the others). This heterogeneity made it difficult
to sequence much more than 40 cycles. Therefore, in order to determine
further amino acids in the sequence, enzymatic cleavage was performed.
Internal sequence of proteins is generally obtained by digesting or cutting
up the large protein molecule into smaller pieces with a combination of
enzymes and chemical cleaveges. When native gelonin was exposed to various
protolytic enzyzme digestions, it was found to be incompletely cleaved.
This was found to be partly due to a disulfide bond in the N-terminal part
of the molecule. Breaking of this bond by reduction and alkylation with
iodoacetic acid yielded a fragment that was less soluable than the native
material at the pH required for enzymatic digestion. A combination of
digestion of native gelonin with trypsin, Lysine aminopeptidase (Lysc),
staphlococcal protease (V8), and chymotrypsin yielded peptides mostly from
the C-terminal portion of the molecule. This indicated that the N-terminal
part of the molecule (from the N-terminal analysis to the Asp-Ala-Pro at
residue 70) was not readily accessible by enzyme digestions.
Gelonin was cleaved with cyanogen bromide into 3 large peptides. Protein
aliquots (0.2 mg/ml) were dissolved in 70% formic acid. A crystal of
cyanogen bromide was added to the solution and the reaction allowed to
proceed for at least 18 hours. The solution was then diluted with water
and was applied to a small sequencing column. After sample application, a
gradient of 1% to 10% n-propanol with 0.1% TFA was used to elute the
protein fragmants. The elution profile is shown on FIG. 4.
Enzymatic digestion of the whole protein or of CNBr fragmants yeilded
overlapping peptides. Enzymatic digestions with Lysyl endopeptidase in
0.1% SDS 100 mM Tris pH 8.0, Staphylococcus Aureus Protease in 0.1% SDS or
trypsin in 0.1% Tween 20 were carried out. Gelonin contains one cysteine
residue at position 49. Reduction and carboxymethylation yeilds a protein
which recovers better on reverse phase HPLC and is more susceptible to
enzymatic digestion. Therefore, most of the enzymatic digestions were
carried out in 0.1% SDS or 0.1% Tween.
After the C-terminal 160 residues were aligned by a combination of CNBr
digests and enzymatic cleaveges. The remaining unknown sequence between
residues 40 to 70 was determined by a combination of chemical modification
of cysteine with iodoacetic acid and solubization of the alkylated protein
with SDS. The RCM alkylated gelonin was then cleaved with excess Lysc
enzyme at 37.degree. C. for short periods of time (1-5 hr.). The HPLC
elution profile is shown on FIG. 5A.
This method yielded a new sequence that had not been seen before. This new
sequence showed the existence of an Asn-Gly combination. This combination
of amino acids is cleaveable by a chemical method using hydroxylamine.
Hydroxylamine cleavage was carried out by adding 100 ug of gelonin to
freshly prepared hydroxylamine (2M) in 0.2 M Tris (pH 9.0) with 2M NaCl,
mm EDTA and 10% ethanol. After incubation for 7 hours at room temperature,
the entire reaction mixture was applied to a sequencing column. The
colulmn was then washed with 1% TFA in water and either eluted with an
acetonitrile gradient or was sequenced directly as a mixture. This
chemical cleavege produced a large hydrophobic peptide that contained
about a 200 amino acid sequence which connected with the Asp-Ala-Pro at
residue 70. The elution profile is shown on FIG. 5C.
The remaining, short section of overlapping sequence from between residues
40 to 50 was determined by digesting gelonin without alkylation by Lysc in
SDS. This digested away most of the C-terminal part of the material. Then
this mixture was digested again by chymotrypsin. The products of this
digestion were then separated by HPLC. Sequence analysis of a large
pepticle revealed a sequence (SerThrLys) starting about 5 amino acids in
from the N terminal end of the molecule. This was useful in that it
removed the heterogeneous part of the molecule and allowed for a longer
sequence run.
Gelonin protein comprises 258 amino acids, the sequence of which is
demonstrated on FIG. 1. The amino acid sequence of gelonin was compared to
other known sequences available in sequence data banks (Genbank, PIR,
EMBL) to determine whether gelonin has any areas of homology with other
proteins. Comparison of the gelonin amino acid sequence with other
proteins having known amino acid sequences, demonstrated that the gelonin
sequence is unique. Homology of certain portions of the gelonin sequence
to portions of other proteins was detected. For instance, gelonin
demonstrates a 36.0% homology with alphatrichosanthin from Trichosanthin
kirilowi, 33.8% homology with Abrin A chain from Indian Licquorice, 35.2%
homology with agglutinin precursor from Castor bean, 33.7% homology with
Ricin D, A chain from Castor bean and 27.3% homology with antiviral
protein (MAP) from Mirabills jalapa. A summary of the degree of homology
to these and other proteins is shown on FIG. 3.
Hydrophobicity plots shown on FIG. 6A-6E demonstrate a similarity to
hydrophobic regions of trichosanthin, Ricin and to other ribosomal
inhibiting proteins.
A plot of the hydropathy of the gelonin structure shows a hydrophobic
region in residues 35-80 and 150-180. These are areas in which substantial
folding of the molecule probably occurs. This similar hydrophobic pattern
is also observed for other toxins (see FIGS. 6A-6E) and may suggest that
the active enzymatic center may be contained within these folded regions.
Therefore, the active enzymatic site may not be found in a linear region
of the molecule and these structures may need to be adequately folded to
attain the proper enzymatic center.
Utilizing the cDNA of gelonin, recombinant gelonin can be produced.
Mutations can be specifically introduced into the molecule in order to
provide recombinant gelonin lacking carbohydrate groups which can
misdirect gelonin-antibody conjugates. Recombinant gelonin molecules can
be produced by site directed mutagenesis to have greater toxic activity
than the native molecule, to be more effectively internalized once bound
to the cell surface by a carrier such as a monoclonal antibody or a
targeting ligand such as IL-2, EGF, IFN, etc., to resist lysosomal
degradation and thus be more stable and longer acting as a toxic moiety.
Recombinant gelonin molecules can also be engineered as fusion products to
contain other functional modalities to kill cells such as an enzymatic
activity, TNF, IFN activity, a second toxic activity, such as diptheria
toxin action (wherein said second activity was through a different
biological pathway than gelonin), thus creating a "supertoxin" or a toxin
with multifunctional actions.
Fusion proteins can be engineered with gelonin to carry drugs such as
chemotherapeutic agents or isotopes for radioimaging or radiotherapy.
Gelonin peptides may have application as abortofacient agents, immuno
suppressive agents, anticancer agents and as antiviral agents (such as an
anti-HIV agent).
The following examples provide a detailed description of the preparation,
characterization, and amino acid sequence of gelonin. The experimental
methods utilized are described in detail in the examples below. These
examples are not intended to limit the invention in any manner.
EXAMPLE 1
Purification and Characterization of Gelonin
Gelonin was isolated from the seeds of the plant Gelonim multiforum
essentially according to the procedure as described (Stirpe, et al. (1980)
J. Biol, Chem 255 6947-6953). Briefly, gelonin was extracted from the
seeds by homogenization in buffered saline solution (pH 7.4). The
supernatant was concentrated after dialysis against 5 mM sodium phosphate
(pH 6.5) and the gelonin further purified by ion exchange chromatography
as described below. The purity of the gelonin toxin was assessed by high
pressure liquid chromatography (HPLC) and sodium
dodecylsulphate-polyacylamide gel electrophoreseis (SDS-Page). Gelonin
toxin migrated as a single band with an approximate molecular weight of
29-30,000 daltons.
Gelonin toxin activity was measured as described in Example 2 by protein
synthesis inhibition in a cell-free system.
Seeds of Gelonium multiflorum were shelled and the nuts ground in a
homogenizer with eight volumes of 0.14 M NaCl containing 5 mM sodium
phosphate (pH 7.4). The homogenate was left overnight at 4.degree. C. with
continuous stirring, cooled on ice and centrifuged at 35,000 times g for
20 minutes at 0.degree. C. The supernatant was removed, dialyzed against 5
mM sodium phosphate (pH 6.5) and concentrated using a pm10 filter. The
sample was layered on a CM-52 ion-exchange column (20.times.1.5 cm)
equilibrated with 5 mM sodium phosphate (pH 6.5). Material which bound to
the ion exchange resin was eluted with 400 ml of 0 to 0.3 M linear NaCl
gradient at a rate of 25 ml hour at 4.degree. C. Five ml fractions were
collected. The fractions were monitored at 280 nm in a spectrophotometer.
The gelonin eluted in about fractions 55-70 and was the last major elution
peak. These fractions were pooled, dialyzed against 0.1 M NaCl in 0.1 M
Na.sub.2 HPO.sub.4 buffer (pH 7.4). The sample was then applied to a
Cibacron blue sepharose column (24.times.2 cm) previously equilibrated
with 0.1 M Na.sub.2 HPO.sub.4 /0.1 M NaCl buffer. The column was washed
with 3 column volumes of buffer and eluted with a 400 ml linear salt
gradient (from 0.1 M NaCl to 2 M NaCl). Elution of the bound material was
monitored by Lowry assay of the column fractions. The fractions containing
the single protein peak were pooled and dialyzed overnight at 4.degree. C.
against PBS. Gelonin toxin was purified to greater than 97% purity as
estimated from silver stained PAGE. The purity and the molecular weight of
each preparation was checked on high pressure liquid chromotography using
a TSK 3000 gel permeation column with 50 mM sodium phosphate buffer, pH
7.4 and 15% sodium dodecylsulphate-polyacrylamide gel electrophoresis
(SDS-page). Gelonin migrated as a single band with an approximate
molecular weight of 29-30,000 daltons.
EXAMPLE 2
Assay of Gelonin Activity
The gelonin activity was monitored in a cell-free protein synthesis
inhibition assay. The cell-free protein synthesis inhibition assay was
performed by sequentially adding to 50 ul rabbit reticulocyte lysate,
thawed immediately before use, mixing after each addition, the following
components: 0.5 ml of 0.2 M Tris HCl (pH 7.8), 8.9 ml of ethylene glycol,
and 0.25 ml of 1 M HCl).
Twenty microliters of a salt-amino acid-energy mixture (SAEM) consisting
of: 0.375 M KCl, 10 mM Mg(CH.sub.3 CO.sub.2).sub.2, 15 mM glucose, 0.25-10
mM amino acids (excluding leucine), 5 mMATP, 1 mMGTP, 50 mMTris-HCl (pH
7.6), 10 ul Creatinine phosphate-creatinine phosphokinase, 8 ul [.sup.14
C] leucine (Amersham, 348 mCi/mmol), and adding 1.5 ul of solutions
containing varying concentrations of the gelonin mixture. The mixture was
incubated for 60 minutes at 30.degree. C. .sup.14 C-leucine incorporation
was monitored in an aliquot of the mixture by precipitating synthesized
protein on glass fiber filters, washing in 10% TCA and acetone, and
monitoring the radioactivity in a Beta-counter using Aquasol scintillation
fluid. Utilizing this assay, purified gelonin had a specific activity of
4.times.10.sup.9 U/mg protein. A unit of gelonin activity is the amount of
gelonin protein which causes 50% inhibition of incorporation of [.sup.14
C] leucine into protein in the cell free assay.
EXAMPLE 3
Determination of Gelonin Amino Acid Sequence
The gelonin amino acid sequence was determined by the Edman degradation
method using an automated amino acid sequencer as described in European
Patent Application No. EP-257735. Large peptides and unfragmented protein
were applied to the reverse phase portion of the sequence reaction
chamber. Unwanted buffer components were washed off with excess water. The
protein or peptide sample was then sequenced by Edman chemistry and the
extracted ATZ amino acid derivatives were converted to the PTH form by 25%
TFA in H.sub.2 O at 65.degree. C. PTH samples were identified by reverse
phase analytical separation on a Np 1090 column.
In order to obtain further amino acid sequence, the protein was digested
with various proteolytic and chemical agents and then the peptides were
purified by high performances liquid chromatography. Gelonin was found
quite resistant to the exposure of trypsin (cleaves after arginine and
lysine residues) and acetyl trypsin (cleaves only after lysine residue).
The protein was found resistant to as much as 5% (w/w) of the enzyme. The
resistance of gelonin to the proteolytic enzyme trypsin is not due to a
lack of trypsin cleavage sites, since gelonin contains 21 lysine and 12
arginine residues. These results indicate that gelonin is perhaps a
rigidly packed molecule which makes it inaccessible to proteolytic
enzymes.
Since gelonin was found resistant to cleavage by proteolytic enzymes,
chemical cleavage of the protein was examined.
EXAMPLE 4
CNBr Cleavage of Gelonin
Gelonin prepared as in Example 1 was dissolved in 70% formic acid. A
crystal of cyanogen bromide was added to the solution. After at least 18
hours the solution was applied to either a small column (0.15 cm.times.5
cm) reverse phase (J. T. Baker; 15 cm C-1B bonded phase Cat II 7191-02) or
analytical (4.6.times.100 mm) reversed phase column. A gradient elution of
1 to 70% n propanol with 1% TFA in water produce 5 peaks as shown on FIG.
6. Each of the peaks were sequenced and also used for further digestion by
enzymes to piece together the entire sequence. Peak 1 was sequenced
directly and gave a sequence starting with a Phe (F) that ran for 38
residues and ending with a Glu (E). This sequence was confirmed by mass
spectroscopy and Lysc digestions of this isolated peptide. Peak 2 was
sequenced directly and gave a sequence starting with a Val (V) that ran
for 47 cy and was not interruptable after the ala at cy 47. Peak 3 was
sequenced and gave the same sequence as peak 2. SDS gels of peaks 2 and 3
as well as Lysc digestion of peaks 2 and 3 showed that peak 3 contained
the C-terminal CNBr peptide as well. Subsequent trypsin digestion of
gelonin produced a peptide that connected these two CNBr peptide
sequences. This trypsin peptide when sequenced gave the sequence
TSGANGMFSEAVELER. Peak 4 and 5 both gave the N-terminal sequence GLDT . .
. . This was used for some digestion by Lysc, 1/8, to give peptides from
its C-terminal end.
EXAMPLE 5
Enzymatic Digestion of CNBr Cleaved Gelonin
Samples of whole protein or CNBr fragments were digested with Lysyl
endopeptidase (Wako Chemical Dallas, Tex.) in 0.1% SDS 100 mmTris pH 8.0
or Staphylococcus Aureus Protease (Pierce) in.1% SDS or Trypsin (Sigma) in
0.1% Tween 20. Digestion mixtures were separated by HPLC and collected
peptides were sequenced on the prototype sequence use gas-phase Edman
sequencing methods.
EXAMPLE 6
Amino Acid Sequence of Gelonin
A total of 258 amino acid residue sequences were obtained following
analysis of the CNBr fragments obtained in Example 3. FIG. 1 shows the
amino acid sequence of gelonin. Gelonin contains a total of approximately
258 amino acid residues. The DNA sequence was deduced from this amino acid
sequence. The degenerate DNA sequence is shown on FIG. 2. Those skilled in
the art will recognize that fragments and derivatives of either the
gelonin amino acid sequence or the DNA sequence coding for gelonin may
inhibit cellular protein synthesis but not bind to a cell surface
receptor.
The invention now being fully described, it will be apparent to one of
ordinary skill in the art that many changes and modifications can be made
thereto without departing from the spirit or scope of the invention as set
forth below.
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